The present invention relates to peptide molecules homing specifically to neovascular tissues and, more particularly, to angiogenesis-specific (neovascular-specific) peptides which function as ligands to neovascular endothelial cells of cancer tissues, for instance, and are useful as molecular drugs and applicable to drug delivery system (DDS) preparations enabling selective drug delivery to target tissues and can contribute to improvements in the therapeutic effects on cancer.
One of the factors which make cancer chemotherapy difficult to do successfully are the fact that the drug administered kills or damages not only the target cancer tissues but also normal tissues, causing adverse effects. As means of minimizing such side effects and achieving improvements in the efficacy of anticancer agents, drug delivery systems (DDSs) have attracted attention in the field of cancer therapy.
The DDSs mentioned above which are targeted at cancer may be classified into two types, namely passive targeting type and active targeting type. Since neovascular tissues show increased vascular permeability as compared with pre-existing vessels, a preparation of the long retention-in-blood type is gradually accumulated in cancer tissues. Passive targeting is the targeting utilizing that property. A passive targeting preparation in which liposomes are used has already been used in Europe and America in the treatment of Kaposi""s sarcoma. On the other hand, an active targeting preparation is designed, by modifying the drug with an antibody or some other ligand capable of binding to a cell surface marker, such as a protein, highly expressed in cancer cells or tissues surrounding the same, so that the drug can be delivered actively and selectively to cancer cells, without causing harmful effects on normal tissues.
In the field of current cancer therapy, angiogenesis has become a focus of attention. The term xe2x80x9cangiogenesisxe2x80x9d refers to the development of blood vessels within cancer tissues which parallel the growth of tumors in the procession of cancer. Thus, for active proliferation of cancer cells and growth and metastasis of cancer tissues, it is important that blood vessels, which are organs serving to feed nutrients and oxygen and eliminate metabolites and waste materials, be newly constructed. In this respect, the growth of cancer tissues can be highly dependent on angiogenesis.
It is considered that when this angiogenesis is inhibited, the growth and metastasis of cancer tissues, might be prevented. From this point of view, it is desired in the art that a cancer therapy targeted on neovascular tissues, in particular an active targeting preparation (DDS preparation), be developed.
A substance capable of serving as a ligand for neovascular endothelial cells in a cancer tissue, if identified, isolated and made available, will lead to its application in DDS preparations and to further improvements in the efficiency of cancer therapy.
It is an object of the present invention to provide such a novel ligand.
Another object of the invention is to provide a substance capable of inhibiting angiogenesis.
In the course of intensive investigations made for the above purposes, the inventors obtained the findings mentioned below. Thus, the inventors first induced the formation of tumor neovascular tissues in the mouse dorsum by the chamber ring method (Folkman, J., et al., J. Exp. Med., 133, 275-288 (1971)). Then, a random peptide-displaying phage constructed by inserting random DNAs into the phage+ coat protein pIII gene to thereby enable the expression of random peptides having a 15-amino-acid sequence on the phage shell was administered to the mice. Thereafter, the mice were frozen with liquid nitrogen, skin portions bearing neovascular tissues were dissected, homogenized in a culture medium containing a protease inhibitor, washed and centrifuged, and the phage was thus recovered from neovascular tissues. The phage was infected into Escherichia coli, which was mass-cultured. After isolation and purification, there was obtained a phage capable of expressing a peptide to be accumulated in the neovascular tissue endothelium and serve as a ligand. For a plurality of phages obtained in that manner, the peptides expressed by them were sequenced.
Then, for selecting a phage expressing a peptide having high affinity for neovascular tissues, each phage obtained in the above manner was administered into the tail vein of tumor-bearing mice prepared by tumor cell implantation. The mice were frozen in the same manner as above, tumor tissues were dissected, and the phages were isolated and purified from the materials obtained and used to infect Escherichia coli, followed by cultivation. And, for each phage, colony-forming units were counted, with the phage before selection being used as a control. The affinity for neovascular tissues was calculated in terms of the ratio of number of phages administered to the tail vein to number of accumulated phages per 100 mg of tumor tissue. In this way, candidate peptides for ligands having high affinity for neovascular tissues were obtained.
Further, the inventors synthesized the above peptides, dendrimers thereof, partial peptides thereof and the like, and confirmed that these peptides actually show antitumor effects and, at the same time, confirmed that liposomes modified with the peptides, in particular these peptides which contains the sequence Trp-Arg-Pro and the sequence Pro-Arg-Pro, show significantly higher levels of distribution in the tumor as compared with the control.
The present invention was accomplished on the basis of these findings.
The invention provides an angiogenesis-specific peptide selectively homing to neovascular tissues, which comprises one of the members listed below under (a) and (b):
(a) a peptide having one of the amino acid sequences shown in SEQ ID NO: 1 to 11, or a dendrimer thereof,
(b) a peptide having an amino acid sequence derived from any of the amino acid sequences of the peptide defined above under (a) by substitution, deletion or addition of one or a plurality of amino acid residues and having affinity for neovascular tissues, or a dendrimer thereof.
In particular, the invention provides an angiogenesis-specific peptide as mentioned above which is a peptide having one of the amino acid sequences shown in SEQ ID NO: 1 to 11, or a dendrimer thereof; more preferably, an angiogenesis-specific peptide as mentioned above which is a peptide having one of the amino acid sequences shown in SEQ ID NO: 1, 5 and 6, or a dendrimer thereof; an angiogenesis-specific peptide as mentioned above which is a dendrimer comprising a plurality of peptides which are the same or different and have one of the amino acid sequences shown in SEQ ID NO: 1 to 11; an angiogenesis-specific peptide as mentioned above which is a peptide having one of the amino acid sequences shown in SEQ ID NO: 12 to 17, or a dendrimer thereof; and an angiogenesis-specific peptide as mentioned above which is a peptide having one of the amino acid sequences shown in SEQ ID NO:19, 21, 23-25 and 28-32, or a dendrimer thereof.
The invention further provides an angiogenesis-specific peptide as mentioned above which homes selectively to neovascular tissues developed in cancer/tumor tissues, for example sarcoma or melanoma.
The invention still further provides an anticancer composition and a cancer metastasis inhibitor composition, each of which comprises, as an active ingredient, at least one of the above angiogenesis-specific peptides, preferably at least one peptide having one of the amino acid sequences shown in SEQ ID NO:1, 5, 6, 13-17, 19, 21, 23-25 and 28-32 or dendrimer thereof, together with a pharmaceutical carrier therefor.
The invention further provides a liposome preparation which comprises, as active ingredients, at least one of the above angiogenesis-specific peptides, preferably at least one peptide having one of the amino acid sequences shown in SEQ ID NO:15-17, or dendrimer thereof, and an anticancer agent or cancer metastasis inhibitor, together with a pharmaceutical carrier therefor.
The invention further provides a method of combating cancer/tumor or inhibiting cancer metastasis which comprises administering an effective amount of at least one of the above angiogenesis-specific peptides to a patient, in particular a method of combating cancer/tumor or inhibiting cancer metastasis which comprises administering an effective amount of at least one peptide having one of the amino acid sequences shown in SEQ ID NO:1, 5, 6, 13-17, 19, 21, 23-25 and 28-32, or at least one dendrimer thereof, to a patient.
The invention further provides a method of combating cancer/tumor or inhibiting cancer metastasis which comprises administering a liposome preparation comprising, as active ingredients, at least one peptide having one of the amino acid sequences shown in SEQ ID NO:15-17, or at least one dendrimer thereof, and an anticancer agent or cancer metastasis inhibitor, together with a pharmaceutical carrier therefor, to a patient.
Hereinafter, the amino acids, peptides, base sequences, nucleotides and the like, when indicated by symbols, are indicated according to the recommendations of the IUPAC-IUB or the xe2x80x9cGuideline for preparing specifications etc. containing nucleotide sequences or amino acid sequencesxe2x80x9d (edited by the Japanese Patent Office) and the conventional symbols used in the relevant field of art.
Specific examples of the angiogenesis-specific peptide of the invention are these having the amino acid sequences shown in SEQ ID NO:1 to 11 which are obtained by the methods shown in the examples given later herein.
In the following, the identification and affinity for neovascular tissues of the angiogenesis-specific peptide of the invention are described.
For identifying the angiogenesis-specific peptide of the invention, the molecular library screening technique can be employed. A preferred example of the library is a phage-displayed library. Such a library may be a commercially available one. The random peptide-displaying phage in said library is utilized for causing expression of a large number of peptides, which can be screened in vitro using a specific target molecule or objective cell, for identifying a peptide specifically binding to the target molecule or cell. The screening using such a library is utilized to identify ligands or various antibodies specifically binding to various cell surface receptors. For the method of constructing such a phage-displayed library and the method of in vitro screening, reference is made to the method of Scott and Smith (Scott, J. M. and Smith, G. P., Science, 249, 386-390 (1990); Smith, G. P. and Scott, J. K., Methods in Enzymology, 217, 228-257 (1993)).
More preferable method to be used in identifying the angiogenesis-specific peptide of the invention as a molecule capable of homing to neovascular tissues is, for example, the method of Ruoslahti et al. described in JP Kohyo H10-502674 (corresponding to U.S. Pat. No. 5,622,699) which identifies a molecule homing to an organ or tissue. The method identifies a molecule homing specifically to one, two or three selected organs or tissues using in vivo panning for screening a library of molecules potentially homing to an organ or organs and can be carried out in the following manner.
Thus, first, random DNAs are introduced into a known phage library, and the thus-obtained diluted mixture of the phage library is administered into the tail vein of a mouse, for instance. One to four minutes later, the mouse is rapidly frozen in liquid nitrogen. For phage recovery, the dead body is thawed, the desired organ or tissue is collected and homogenized in a culture medium containing a protease inhibitor and the preparation obtained is washed several times with an ice-cooled culture medium containing 1% bovine serum albumin and used to infect Escherichia coli. The phage-infected Escherichia coli is cultured in a tetracycline-containing medium for several hours and then used to precoat a tetracycline-containing agar plate. The phage-containing colonies recovered are cultured on an appropriate medium, and phages are isolated and purified. And, the second and subsequent biopanning procedures are carried out. This second, and subsequent, biopanning can be carried out in the same manner as mentioned above using the phages obtained in the above manner. Thus, a DNA coding for a peptide expressed by a desired and selected phage can be obtained. By sequencing the DNA obtained, the molecule homing to the desired organ or tissue can be identified.
The DNA sequencing can be readily carried out by a method well known in the art, for example by the dideoxy method [Proc. Natl. Acad. Sci. USA, 74, 5463-5467 (1977)] or the Maxam-Gilbert method [Methods in Enzymology, 65, 499 (1980)]. Such base sequence determination can also be carried out with ease using a commercial sequencing kit or the like.
As a method of detecting the affinity of a molecule homing to an organ or tissue, the following method, for instance, can be employed. Thus, in the above-mentioned method of identifying a molecule homing specifically to an organ or tissue, the organ- or tissue-specific peptide-expressing phage obtained and the phage before selection are administered into the tail vein of experimental animals, and phage-containing colony forming units are counted by the same method as mentioned above. By evaluating the ratio of number of accumulated phages to number of phages administered into the tail vein per 100 mg of the target organ or tissue, for instance, the affinity of the molecule homing to the desired organ or tissue can be detected.
Further, the homing specificity of a peptide can be confirmed by the competitive method, for instance, by selecting one of peptides homing to the target organ or tissue, synthesizing said peptide, purifying the same by high performance liquid chromatography (HPLC) and examining the effects of several peptide phages containing a phage expressing the same peptide as the above synthetic peptide and homing to the target organ or tissue by simultaneous administration with the synthetic peptide (cf. JP Kohyo H10-502674; U.S. Pat. No. 6,522,699).
The details of the methods of identifying the angiogenesis-specific peptide of the invention and detecting the affinity for neovascular tissues thereof are as shown later herein in the examples. The angiogenesis-specific peptide of the invention as identified by in vivo panning in mice as shown later in an example can bind to the neovascular tissues of solid tumors in human or other mammalian species. The peptide of the invention which binds to a target molecule occurring in the neovascular tissue grown in mice can bind to the corresponding molecule in the neovascular tissues of tumors in human or other mammalian bodies. Further, the peptide of the invention can specifically bind in vitro to a sample obtained from a patient. From these facts, it can be confirmed that the peptide of the invention has the ability to bind to the corresponding molecule of the human patient.
The vascularization in cancer tissues is characterized in that the formation of new blood vessels supporting the growth occurs continuously; it is thus distinguished from ordinary histological vascularization (Folkman, Nat. Med., 1, 27-31 (1995); Rak, Anticancer Drugs, 6, 3-18 (1995)). Therefore, the peptide of the invention specifically homing to neovascular tissues as identified by in vivo panning can be used as an angiogenesis-inhibiting factor against cancer.
On the other hand, the peptide of the invention, which specifically homes to neovascular tissues, is very low in the possibility of producing adverse effects on normal healthy organs and tissues.
Further, since the peptide of the invention homes not to cancer cells but to the neovascular tissue, the possibility of its acquiring drug resistance such as the case with anticancer agents is considered to be low.
The peptide of the invention which homes specifically to neovascular tissues can further be used targeting other new blood vessels such as those occurring in inflammatory tissues or regenerated or injured tissues. Further, neovascularization occurs in uterine tissues as well, and the peptide of the invention is considered to be able to bind to such uterine tissues and is expected to exert an influence on such diseases as hysteromyoma.
The peptide of the invention as identified and established in the above manner includes the peptides specified by SEQ ID NO:1 to 11, and these are all characterized by having the property of homing to neovascular tissues.
The peptide of the invention includes peptides having one of the amino acid sequences shown in SEQ ID NO:1 to 11 as well as peptides comprising an amino acid sequence derived from said amino acid sequences by modification through substitution, deletion or addition of one or a plurality of amino acid residues and having affinity for neovascular tissues, namely the property of homing to neovascular tissues.
The extent and positions) of xe2x80x9csubstitution, deletion or additionxe2x80x9d of an amino acid(s) are not restricted provided that the modified proteins are equivalents having the same properties as the angiogenesis-specific peptides respectively comprising the amino acid sequences shown in SEQ ID NO:1 to 11. While the above amino acid sequence modification (mutation) or the like may occur naturally, for example upon mutation or posttranslational modification, artificial modification based on the nature-derived gene is also possible. The invention includes all modified peptides having the above characteristics, irrespective of cause or means of such modification/mutation.
The peptide of the invention further includes homologs of the peptides having the amino acid sequences shown in SEQ ID NO:1 to 11. The homologs include mammalian proteins, for example proteins of the human, horse, sheep, cattle, dog, monkey, cat, bear or rodent (e.g. rat, rabbit) origin, which have the same activities as the peptides having the amino acid sequences shown in SEQ ID NO:1 to 11.
Examples of the peptide of the invention which have an modified amino acid sequences are these having sequences derived from the sequences shown in SEQ ID NO:1, 5 and 6 by allowing the sequences occurring therein overlapping in part, for example Pro-Arg-Pro and Trp-Arg-Pro, to remain and substituting, for an amino acid residue or residues of the remaining amino acid sequence, another amino acid residue or other amino acid residues, deleting an amino acid residue or residues or adding some other amino acid residue or residues; these having sequences derived from the sequence shown in SEQ ID NO:2 by substituting other amino acid resides for the 2nd and 8th amino acid residues; and these having sequences derived from the sequence shown in SEQ ID NO:11 by allowing the amino acid sequence portion from the 4th to the 11th amino acid residue alone to remain and deleting the remaining residues.
Specific examples of the peptide of the invention as derived by partial modification of one of the amino acid sequences are the peptide comprising 12 amino acid residues as shown in SEQ ID NO:19; the peptides comprising 8 amino acid residues as shown in SEQ ID NO:12 to 14 and 21; the peptides comprising 5 amino acid residues as shown in SEQ ID NO:15 to 17 and 23-25; the peptides comprising 4 amino acid residues as shown in SEQ ID NO:28 to 31; and the peptide comprising 3 amino acid residues (Trp-Arg-Pro) as shown in SEQ ID NO:32.
More specifically, the peptide shown in SEQ ID NO:12, for instance, is derived from SEQ ID NO:11 by retaining only the portion from the 4th to 11th amino acid residue. The peptide shown in SEQ ID NO:13 is derived from the sequence comprising 15 amino acid resides as shown in SEQ ID NO:5 by retaining 8 amino acid residues from the N terminus and deleting the remaining 7 amino acid residues. The peptide shown in SEQ ID NO:14 has a sequence of 8 amino acid residues as a result of deletion of the 7 amino acid residues from the N terminus of the amino acid sequence shown in SEQ ID NO:6. The peptide shown in SEQ ID NO:15 has the sequence from the 2nd to the 6th amino acid residues of the amino acid sequence shown in SEQ ID NO:5. The peptide shown in SEQ ID NO:16 has the sequence from the 9th to the 13th amino acid residues of the amino acid sequence shown in SEQ ID NO:6. The peptide shown in SEQ ID NO:17 is derived from the 1st to 4th amino acid residues of the amino acid sequence shown in SEQ ID NO:1 by addition of Ala to the N terminus thereof.
Among the peptides which the present invention includes, peptides having at least two cysteine residues, for example the peptide having the amino acid sequence shown in SEQ ID NO:11, are considered to spontaneously cyclize, and such cyclic peptides are also active in some instances even when they occur in the linear form and, therefore, one or both of the cysteine residues in said peptides do not exert a significant influence on the homing characteristic of the peptides, hence can be deleted. Such phenomenon is supported, for example, by the report by Koivunen et al. (J. Biol. Chem., 268, 20205-20210 (1993)). A specific example of such peptide having an amino acid sequence resulting from deletion is as shown in SEQ ID NO:12. Peptides having such a partly deleted amino acid sequence, too, if they have the above-mentioned property of homing to neovascular tissues, fall within the scope of the invention.
As used herein, the term xe2x80x9cangiogenesis-specific peptidexe2x80x9d or xe2x80x9cpeptide of the inventionxe2x80x9d includes peptides having a modified amino acid sequence derived from any of the above-mentioned amino acid sequences as shown in SEQ ID NO:1 to 11 as standards, for example partial peptides derived therefrom by deletion of a partial amino acid sequence.
The angiogenesis-specific peptide of the invention includes peptides having one of the amino acid sequences shown in SEQ ID NO:1 to 11, peptides whose amino acid sequence is derived from said amino acid sequences by modification and which has the property of homing to neovascular tissues, and further dendrimers of these peptides.
The term xe2x80x9cdendrimerxe2x80x9d is used herein to mean a peptide known as a macromolecule having a specific composition, a specific molecular weight, and a spherical or three-dimensional structure and also known as a multiple antigen peptide (MAP). The synthesis thereof can be carried out, for example, starting with a chemical structure nucleus having a plurality of functional groups, causing a branch (repeating unit) terminally having a plurality of the same functional groups as these of the chemical structure nucleus to be bound to each functional group of the nucleus and further introducing the same repeating unit one by one into the terminal functional groups. The details are described, for example, in JP Kohyo S60-500295, JP Kokai S63-99233, JP Kokai H03-263431, U.S. Pat. No. 4,507,466, U.S. Pat. No. 4,568,737, Poloymer Journal, vol. 17, page 117 (1985), Tomalia, et al., Angewandte Chem. Int. Engl., vol. 29, pages 138-175 (1990), and Macromolecules, vol. 25, page 3247 (1992).
Dendrimers as mentioned above comprise a core moiety serving as a starting nucleus for a spherical appearance with a branched or stellar configuration, internal layers (generations of ramifications) constituted of repeating units radially extend outwardly from the starting core, and an external surface comprising activated functional groups bound to respective outermost termini of respective generations or branches. The size, shape and reactivity of a dendrimer can be adjusted by selecting the starting core moiety, the generation of the dendrimer and the composition and structure of the repeating unit to be used for each generation.
Dendrimers differing in size can be obtained by increasing the generations employed and, for their production, reference may be made to U.S. Pat. No. 4,694,064, for instance.
Typical dendrimers include, for example, dendrimers comprising a nitrogen atom as the core moiety serving the starting nucleus, repeating units having the structure xe2x80x94CH2CH2CONHCH2CH2xe2x80x94 bound to the core and activated functional groups which are bound to the outermost terminal amino groups of each branches and whose constituents are angiogenesis-specific peptides of the invention as shown in SEQ ID NO:1 to 17; and dendrimers shown later in the examples which comprise an amino acid, such as lysine, arginine, glutamic acid or aspartic acid, as the core moiety, the same amino acids as mentioned above as the repeating units directly bound to the core moiety, and angiogenesis-specific peptides of the invention having an amino acid sequence selected from among SEQ ID NO:1 to 17 or angiogenesis-specific peptides of the invention having an amino acid sequence selected from among SEQ ID NO:19, 21, 23-25 and 28-32 as the activated functional groups.
Dendrimers with an angiogenesis-specific peptide of the invention bound to the outermost terminus of each branch can be produced by the solid-phase synthetic method described later herein using a dendrimer having a nitrogen atom serving as the starting core moiety, which is commercially available from Polysciences, Inc., 400 Vally Road, Warrington, Pa., 18976, U.S.A., for instance. Similarly, dendrimers comprising angiogenesis-specific peptides of the invention bound to the outermost terminus of each branch can be produced by the solid-phase synthetic method described later herein using lysine as the core moiety serving as the starting site, and the same amino acid lysine as the repeating unit directly bound to the core moiety. In producing the dendrimer, a Fmoc8-Lys4-Lys2-Lys-xcex2-Ala-Alko resin produced by Watanabe Kagaku Kogyo can be used. In the above process, it is also possible to produce dendrimers containing constituents having anticancer activity either in lieu of part of the angiogenesis-specific peptides of the invention to be bound to the outermost terminus of each branch or in the form bound to the core moiety.
The dendrimers mentioned above each can be synthesized, for example, in the following manner. Thus, dendrimers can be obtained by condensing a resin for solid-phase peptide synthesis, via a spacer or without any spacer, with an xcex1,xcfx89-diamino acid, as a repeating unit, wherein two amino groups are protected with the same or different protective groups, followed by deprotection and by repetitions of the condensation of the repeating unit, each time followed by deprotection.
Usable as the resin for solid-phase peptide synthesis are resins generally used in peptide synthesis, such as polystyrene, polyacrylamide, polystyrene-polyethylene glycol and like resins. These resins are used with terminally additional groups of a chloromethyl, 4-(hydroxymethyl)phenoxy, 4-((xcex1-2xe2x80x2,4xe2x80x2-dimethoxyphenyl)-9-fluorenylmethoxycarbonylaminomethyl)phenoxy or the like.
As the spacer, one amino acid or a plurality of amino acids can be used. Examples of the xcex1,xcfx89-diamino acids are lysine, ornithine, 1,4-diaminobutyric acid, 1,3-diaminopropionic acid and the like. The protective groups include a Boc group, an Fmoc group, a Z group and the like. Therefore, the functional groups are an amino group, a carboxyl group, a hydroxy group and the like. When the procedure comprising repeating unit condensation and deprotection is repeated n times, the number of branches becomes 2n. Specific number of the branches is 2 to 16.
Such dendrimers can be purified by ordinary techniques, for example by a chromatographic procedure using a resin capable of size exclusion in a matrix form, such as Sephacryl S-300 (product of Pharmacia), for instance.
The dendrimer peptide thus obtained selectively homes to neovascular tissues proper owing to the occurrence of the angiogenesis-specific peptide of the invention in its branch moieties and produces an angiogenesis inhibiting effect, whereby the desired anticancer effect and cancer metastasis preventing effect can be produced. When it is administered with a known agent having anticancer activity packed therewithin, the dendrimer peptide can allow the agent to act on the target angiogenic site alone, hence it is advantageous in that it can render the anticancer agent less capable of producing side effects.
The angiogenesis-specific peptide to be present in the branch moieties of the above dendrimer peptide are not always one and the same peptide for each branch but may include a plurality of peptides differing in amino acid sequence. As examples, there may be mentioned the combined binding, to different branch moieties, of two or more of the peptides respectively having the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:5 and SEQ ID NO:6, or of two or more of the peptides respectively having the 15 amino acid sequences shown in SEQ ID NO:1-11, the peptides respectively having the 8 amino acid sequences shown in SEQ ID NO:12-14 and 21, the peptides respectively having the 5 amino acid sequences shown in SEQ ID NO:15-17 and 23-25, the peptides respectively having the 4 amino acid sequences shown in SEQ ID NO:28-31, and the peptide having the 3 amino acid sequence shown in SEQ ID NO:32. Such dendrimers can show improved stability in the blood and tissues of the administration target, improved specific activity of each bound molecule, and the like.
The angiogenesis-specific peptide of the invention can be synthesized by a common chemical synthetic method based on the amino acid sequence thereof. Said method includes liquid-phase and solid-phase methods of peptide synthesis. More detailedly, such methods of peptide synthesis include the stepwise elongation technique effecting chain extension using amino acids one by one based on the amino acid sequence information, and the fragment condensation technique comprising synthesizing fragments composed of several amino acids in advance and then coupling the fragments together. Either technique can be used in synthesizing the angiogenesis-specific peptide of the invention.
The method of condensation for use in the above peptide synthesis may be any of various known methods. The specific examples are the azide method, mixed acid anhydride method, DCC method, activated ester method, oxidation/reduction method, DPPA (diphenylphosphoryl azide) method, DCC+additive (1-hydroxybenzotriazole, N-hydroxy-succinimide, N-hydroxy-5-norbornene-2,3-dicarboximide or the like) method and Woodward method. The solvent to be used in each of these methods can adequately be selected from among those ordinary ones which are well known to be useful in this kind of peptide condensation reaction. Examples of the solvents include N-methylpyrrolidone (NMP), dimethylformamide (DMF), dimethyl sulfoxide (DMSO), hexamethylphosphoramide, dioxane, tetrahydrofuran (THF), ethyl acetate, etc., and mixture thereof.
In the peptide synthesis reaction, the carboxyl group of an amino acid or peptide not involved in the reaction can be protected generally by esterification, for example as a lower alkyl ester, such as methyl ester, ethyl ester or tert-butyl ester, an aralkyl ester such as benzyl ester, p-methoxybenzyl ester or p-nitrobenzyl ester, or the like. As for the amino acids having a functional group on the side chain, the hydroxyl group of Tyr, for instance may be protected with an acetyl, benzyl, benzyloxycarbonyl, tert-butyl or like group; such protection is not always necessary, however. Further, the guanidino group of Arg, for instance can be protected with an appropriate protective group such as nitro, tosyl, 2-methoxybenzene-sulfonyl, methylene-2-sulfonyl, benzyloxycarbonyl, isobornyloxycarbonyl or adamantyloxycarbonyl. The elimination reaction of such protective groups in the amino acids, peptides and final product angiogenesis-specific peptide of the invention which have those protective groups can be carried out by conventional methods, for example by catalytic reduction or using liquid ammonia/sodium, hydrogen fluoride, hydrogen bromide, hydrogen chloride, trifluoroacetic acid, acetic acid, formic acid, methane-sulfonic acid or the like.
The thus-obtained angiogenesis-specific peptide of the invention can appropriately be purified by methods generally used in the field of peptide chemistry, for example by ion exchange resins, partition chromatography, gel chromatography, affinity chromatography, high performance liquid chromatography (HPLC), countercurrent distribution or the like.
The angiogenesis-specific peptides of the invention obtainable in the above manner (inclusive of dendrimers thereof; hereinafter the same shall apply) have the ability to specifically home to neovascular tissues and are themselves useful as angiogenesis inhibitors. Further, the angiogenesis-specific peptides of the invention have the ability to specifically home to neovascular tissues of cancer tissues and therefore can be used as ligands for cancer tissues, for example in combination with anticancer agents, such as cancer chemotherapeutic agents, bound thereto.
Examples of various target cancer/tumor diseases include melanoma, carcinoma of colon and rectum, ovarian cancer, liver cancer, mammary cancer, brain tumor, renal cancer, pancreatic cancer, cervix cancer, esophageal cancer, lung cancer, gastric cancer and the like.
As the anticancer agents or components having anticancer activity which can be used as agents in combination with the angiogenesis-specific peptides of the invention, the following various cancer chemotherapeutic agents, inclusive of 5-fluorouracil (5-FU) are exemplified. Thus, there may be mentioned alkylating agents such as cyclophosphamide, melphalan, ranimustine, ifosfamide, nitrogen mustard N-oxide hydrochloride, etc.; metabolic antagonists such as 6-mercaptopurine, ribosides, enocitabine, carmofur, cytarabine, cytarabine ocfosfate, tegafur, 5-FU, doxifluuridine, doxifluridine, hydroxycarbamide, fluorouracil, methotrexate, mercaptopurine, etc.; antitumor antibiotics such as actinomycin D, aclarubicin hydrochloride, idarubicin hydrochloride, epirubicin hydrochloride, doxorubicin hydrochloride, daunorubicin hydrochloride, pirarubicin hydrochloride, bleomycin hydrochloride, zinostatin stymalamer, bleomycin sulfate, mitomycin C, neocarzinostatin, peplomycin sulfate, etc.; antitumor botanical preparations such as etoposide, irinotecan hydrochloride, docetaxel hydrate, vincristine sulfate, vindesine sulfate, vinblastine sulfate, paclitaxel, etc. and, further, aceglatone, ubenimex, cisplatin, sizofiran, sobuzoxane, krestin, toremifene citrate, medroxy-progesterone acetate, tamoxifen citrate, carboplatin, fadrozole hydrochloride hydrate, procarbazine hydrochloride, mitoxantrone hydrochloride, L-asparaginase, tretinoin, nedaplatin, picibanil, flutamide, pentostatin, porfimer sodium, lentinan, etc.
Examples of the cytokines having antitumor activity are IFN-xcex1, IFN-xcex2, IFN-xcex3, IL-1, IL-2, IL-12, TNF, TGF-xcex2, angiostatin, thrombospondin, endostatin, etc. Examples of the antibodies or antibody fragments are antibodies or antibody fragments against factors involved in the growth and promotion of cancer, such as anti-VEGF antibody, anti-FGF antibody, anti-HGF antibody I and anti-L-8 antibody.
Thus, the angiogenesis-specific peptide of the invention can be used in producing DDS preparations, for example by coupling or modifying an active agent, such as an antineoplastic agent or a cytokine having anticancer activity, with the same and making up the product into a liposome preparation, and such preparation can be used in active targeting at cancer.
When the angiogenesis-specific peptide of the invention is to be coupled with a protein, such as a cytokine, having anticancer activity, the coupling product can be caused to be expressed as a fused protein composed of the peptide of the invention and the cytokine or the like by using the recombinant DNA technology. The production and expression of such fused protein can be realized by the conventional technology in the art. Thus, the fused protein can be prepared by the ordinary recombinant DNA technology (cf. e.g. Science, 224, 1431 (1984); Biochem. Biophys. Res. Comm., 130, 692 (1985); Proc. Natl. Acad. Sci. USA, 80, 5990 (1983)). In the production and expression of such fused protein, the method of Ohno et al. xe2x80x9cTanpaku Jikken Purotokoru 1 Kino Kaiseki Hen, Saibokogaku Bessatsu, Jikken Purotokoru Sirizu (Protein Experiment Protocols Book 1, Function Analyses, Supplement to Cell Engineering, Experiment Protocols Series), 1997, Shujunshaxe2x80x9d can be referred to.
The recombinant fused protein obtained can be isolated and purified, if desired, by any of various separation procedures utilizing the physical, chemical and other properties thereof [cf. e.g. xe2x80x9cSeikagaku Deta Bukku II (Biochemistry Data Book II)xe2x80x9d, pages 1175-1259, 1st edition, 1st printing, published Jun. 23, 1980 by Tokyo Kagaku Dojin; Biochemistry, 25 (25), 8274-8277 (1986); Eur. J. Biochem., 163, 313-321 (1987)]. Said methods specifically include, for example, ordinary reconstitution treatment, treatment with a protein precipitating agent (salting out), centrifugation, osmotic shock procedure, ultrasonic disruption, ultrafiltration, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, high performance liquid chromatography (HPLC), other various liquid chromatographic techniques, dialysis, and combinations thereof. Particularly preferred among the above methods is affinity chromatography using a column with the desired protein bound thereto.
When the angiogenesis-specific peptide of the invention is to be utilized as a ligand by coupling the same with a physical, chemical or biological substance, the physical, chemical or biological substance can be a drug delivery system substance such as a microdevice having cellules capable of containing the above-mentioned cancer chemotherapeutic agent (e.g. anticancer agent). Examples of such drug delivery system substance include liposomes, microcapsules having a permeable or semipermeable membrane, other microdevices having cellules and like biological substances. These substances are generally nontoxic and preferably biodegradable.
The method of coupling one of the above-mentioned various drug delivery system substances capable of containing an agent such as an anticancer agent with the peptide of the invention is well known in the relevant field of art. Specifically, the coupling is carried out by the method of Harlow or Hermanson (Harlow and Lane, Antibodies: A Laboratory Mannual, Cold Spring Harbor Laboratory Press (1988); Hermanson, Bioconjugate Techniques, Academic Press (1996)).
In the following, a liposome preparation is described in detail, as a typical example of the preparation resulting from coupling of the above-mentioned drug delivery system substance with the peptide of the invention.
The liposome preparation is obtained by causing liposomes, which comprises an acidic phospholipid as a membrane constituent or a neutral phospholipid and an acidic phospholipid as membrane constituents, to hold the peptide of the invention.
The acidic phospholipid as a membrane constituent is defined more narrowly than ordinary acidic phospholipids and specifically includes natural or synthetic phosphatidylglycerols (PGs) such as dilauroylphosphatidylglycerol (DLPG), dimyristoylphosphatidylglycerol (DMPG), dipalmitoylphosphatidylglycerol (DPPG), distearoylphosphatidylglycerol (DSPG), dioleoylphosphatidylglycerol (DOPG), yolk phosphatidylglycerol (yolk PG) and hydrogenated yold phosphatidylglycerol as well as natural or synthetic phosphatidylinositols (PIs) such as dilauroylphosphatidylinositol (DLPI), dimyristoylphosphatidylinositol (DMPI), dipalmitoylphosphatidylinositol (DPPI), distearoylphosphatidylinositol (DSPI), dioleoylphosphatidylinositol (DOPI), soybean phosphatidylinositol (soybean PI) and hydrogenated soybean phosphatidylinositol. These may be used singly or two or more of them may be used in admixture.
Examples of the neutral phospholipid are natural or synthetic phosphatidylcholines (PCs) such as soybean phosphatidylcholine, yolk phosphatidylcholine, hydrogenated soybean phosphatidylcholine, hydrogenated yolk phosphatidylcholine, dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), dilauroylphosphatidylcholine (DLPC), distearoylphosphatidylcholine (DSPC), myristoylpalmitoylphosphatidylcholine (MPPC), palmitoylstearoylphosphatidylcholine (PSPC) and dioleoylphosphatidylcholine (DOPC), natural or synthetic phosphatidylethanolamines (PEs) such as soybean phosphatidylethanolamine, yold phosphatidylethanolamine, hydrogenated soybean phosphatidylethanolamine, hydrogenated yold phosphatidylethanolamine, dimyristoylphosphatidylethanolamine (DMPE), dipalmitoylphosphatidylethanolamine (DPPE), dilauroylphosphatidylethanolamine (DLPE), distearoylphosphatidylethanolamine (DSPE), myristoylpalmitoylphosphatidylethanolamine (MPPE), palmitoylstearoylphosphatidylethanolamine (PSPE), dioleoylphosphatidylethanolamine (DOPE), and the like. These may be used singly or two or more of them may be used in admixture.
The liposome membrane mentioned above is formed by a conventional method using the above acidic phospholipid as a single constituent or the above neutral and acidic phospholipids combinedly. Recommendably, the acidic phospholipid is used in an amount of about 0.1 to about 100 mole percent, preferably about 1 to about 90 mole percent, more preferably about 10 to about 50 mole percent, based on the liposome membrane constituents.
In preparing the above liposomes, cholesterol and/or the like may further be added. When cholesterol is added, the fluidity of phospholipids can be adjusted, whereby liposomes can be prepared more expediently. Generally, said cholesterol is added and incorporated in an amount up to the equal amount to the phospholipids, preferably half of to equal to the amount thereof.
The proportions of the active ingredient and acidic phospholipid in a liposome dispersion are recommendably such that the acidic phospholipid accounts for about 0.5 to about 100 equivalents, preferably about 1 to about 60 equivalents, more preferably about 1.5 to about 20 equivalents, relative to the active ingredient.
The amount of the peptide to be used in peptide modification according to the invention in the whole lipid, as expressed in terms of mole percent, may be several mole percent to few-score mole percent, preferably about 5 to about 10 mole percent, generally about 5 mole percent. When the peptide of the invention itself has anticancer activity, as shown later herein in Example 4, the amount may be about 5 to about 40 mole percent. For a water-soluble anticancer agent or a water soluble substance having anticancer activity, which is included in the water phase within liposomes, it is included with an efficiency of 10% to 90%. On the contrary, a liposoluble anticancer agent or a liposoluble substance having anticancer activity can be included with a high inclusion efficiency close to 100% when the desired component is included within the liposome membrane.
The method of producing the above liposomes is now described. In producing said liposomes, various known methods can be used. For example, the liposome membrane constituent is dissolved in an organic solvent such as chloroform, then the solvent is distilled off under reduced pressure to cause formation of a lipid film, an aqueous phase with the agent dissolved therein is added thereto, followed by warming to a temperature above the phase transition temperature of the lipid and further by vortex treatment, homogenization or like treatment, whereby a liposome dispersion is prepared. It is also possible to prepare a liposome dispersion by warming a powdery liposome membrane constituent to a temperature above the phase transition temperature and mixing the same with an aqueous solution of the agent with stirring. The aqueous agent solution to be added may be any one provided that the agent remains dissolved therein, and the level of addition of the aqueous agent solution can also arbitrarily increased or decreased.
If necessary, the particle size distribution of the thus-obtained liposome dispersion can be controlled by ultrafiltration, for example by using a polycarbonate membrane filter. It is also possible to concentrate the dispersion using a dialysis membrane.
In the liposome dispersion, there may be incorporated, as an additive or additives necessary from the preparation designing viewpoint, one or more of various substances such as preservatives, isotonizing agents, buffers, stabilizers, solubilizers and absorption promoters, or the liposome dispersion may be diluted with a solution containing these or water, when necessary. Specific examples of the above-mentioned additives are such preservatives as benzalkonium chloride, benzethonium chloride, chlorhexidine, parabens (e.g. methylparaben, ethylparaben), thimerosal and like preservatives effective against fungi and bacteria; isotonizing agents such as D-mannitol, D-sorbitol, D-xylitol, glycerol, glucose, mannetose, sucrose, propylene glycol, like polyhydric alcohols, sodium chloride and other electrolytes; stabilizers such as tocopherol, butylated hydroxyanisole, butylated hydroxytoluene, ethylenediaminetetraacetic acid (EDTA) and cysteine, and the like.
Specific examples of the liposome dispersion are shown later herein in Examples 5, 7 and 8.
Furthermore, the angiogenesis-specific peptide of the invention can be utilized as a cancer diagnostic agent or the like by utilizing its ability to home specifically to cancer neovascular tissues by coupling therewith a radioactive compound, fluorescent substance, enzyme, biotin, contrast agent, etc. and performing active targeting at cancer.
Further, the angiogenesis-specific peptide of the invention can be used in active targeting at cancer as a pharmaceutical composition comprising, together with an anticancer agent or a cytokine having anticancer activity, liposomes or a lipid emulsion containing said peptide in a form bound to a fatty acid (e.g. behenic acid, stearic acid, palmitic acid, myristic acid, oleic acid), an alkyl group, a cholesteryl group or the like. The details of the production of liposome preparations such as mentioned above are described, for example, in the reference by Woodle et al. (Long Circulating Liposomes: Old drugs, New therapeutics, M. C. Woodle, G. Storm, Eds., Springer-Verlag Berlin (1998)). The details of the production of pharmaa ceutical compositions containing such a lipid emulsion as mentioned above together with an anticancer agent or a cytokine having anticancer activity are described in the reference by Namba et al. (Liposomal applications to cancer therapy, Y. Namba, N. Oku, J. Bioact. Compat. Polymers, 8, 158-177 (1993)).
The angiogenesis-specific peptide of the invention can also be utilized in cancer diagnosis by binding thereto one of various fatty acids, alkyl groups, cholesteryl group and so forth and making the binding product into liposomes or a lipid emulsion containing the same and further coupling therewith a radioactive compound or a contrast agent for contrasting cancer and performing active targeting at cancer using the resulting product. Thus, the peptide can serve as a cancer diagnosing agent for verifying the presence of cancer. The utilization of such diagnostic agent is advantageous particularly in that initial stage tumor and metastatic lesions, which may not be detected by other methods, can be identified. Therefore, the present invention provides a method of cancer diagnosis, in particular a diagnostic method of identifying initial stage cancer and metastatic lesions, as well.
Once the occurrence of cancer has thus been established, it becomes possible, in accordance with another aspect of the present invention, to couple the angiogenesis-specific peptide with an anticancer agent, for example a cancer chemotherapeutic agent, or with a microdevice containing a cancer chemotherapeutic agent or some other anticancer factor to thereby cause the agent to home to the cancer; thus, it becomes possible to perform the desired active targeting, namely selectively killing cancer or cancer cells while reducing the effect on normal tissues or normal cells. In this respect, the present invention provides a method for the treatment of cancer or cancer metastasis and the inhibition of cancer metastasis as well.
The angiogenesis inhibitor or cancer treatment composition of the invention is administered to patients in the form of a preparation composition containing, as the active ingredient, the angiogenesis-specific peptide or a composite thereof with another anticancer agent or the like, together with a pharmaceutically acceptable carrier.
The pharmaceutically acceptable carrier to be used can be suitably selected from among these well known in the art depending on the form of the preparation composition to be prepared. For example, when the composition is to be prepared in the form of an aqueous solution, water or a physiological buffer solution can be used as the carrier. When the composition is to be prepared in the form of an appropriate solution, glycol, glycerol, olive oil or a like injectable organic ester, for instance, may be used as the carrier.
Further, in cases where the above-mentioned composite is to be used as the active ingredient, a compound serving to stabilize or enhance the absorption of the composite, for instance, may be used. Such compounds include carbohydrates such as glucose, sucrose and dextran; antioxidants such as ascorbic acid and glutathione; chelating agents; and stabilizers or excipients such as low molecular proteins and albumin.
The content of the active ingredient in the angiogenesis inhibitor or cancer therapeutic composition (preparation) of the invention is not particularly restricted but can be selected from within a wide range. When the angiogenesis-specific peptide of the invention is used singly as the active ingredient, it is generally desirable that the content thereof in the preparation be selected within the range of about 0.00001 to about 70% by weight, preferably about 0.0001 to about 5% by weight. The dose of the above preparation is not particularly restricted, either, but can be selected within a broad range according to the desired therapeutic effect, method of administration (route of administration), treatment period, age and sex of the patient and other conditions, among others. Generally, the dose is judiciously selected within the range of about 0.01 xcexcg to about 10 mg, preferably about 0.1 xcexcg to about 1 mg, per kilogram of patient""s body weight per day. The preparation may be administered once daily or in several divided doses per day.
The dose of the angiogenesis inhibitor or cancer treatment composition of the invention which is prepared by using the angiogenesis-specific peptide of the invention coupled to an anticancer agent and/or a cancer metastasis inhibitor can suitably be determined depending on the amount of the cancer chemotherapeutic agent (agent) required to produce the desired anticancer effect, for instance. When, for example 5-fluorouracil (5-FU), which is generally used as an anticancer active agent in the clinical application of this kind, is used, said 5-FU is administered generally at a daily dose of about 0.1 mg/kg to about 50 mg/kg. It can be readily understood by the person skilled in the art that the dose of the angiogenesis-specific peptide of the invention which is utilized in a form bound thereto is by itself evident and that such dose can be regarded as the effective dose of the peptide of the invention. Furthermore, considering that the pharmaceutical composition of the invention is characterized by the ability to specifically home to cancer neovascular tissues, it is anticipated that even when the dose of the cancer chemotherapeutic agent is considerably low as compared with the clinical dose in conventional use, remarkable effects will be produced.
As mentioned above, the angiogenesis-specific polypeptide of the invention can be bound to a radioactive compound, a contrast medium or the like for cancer imaging to give a diagnostic agent, and active targeting at cancer can be conducted using that agent. The angiogenesis-specific polypeptide of the invention can also be used in detecting the occurrence of angiogenesis in cells, tissues, organs or parts thereof as isolated from the human body. By such use, the presence of cancer in a sample isolated from the human body can be detected as a result of the fact that the neovascular tissues are ones formed by cancer. The above human sample may be a tissue section or sample obtained by biopsy, or a cell population existing in a tissue culture or adapted thereto. The human sample may be one treated by homogenization, and this is preferred.